1. Field of the Invention
The present invention is related to systems for detecting target ions in a sample, and more specifically, to ion sensors comprising an ionophore covalently anchored into a plasticizer free polymer. This invention is further related to imprinted plasticizer-free polymers for detecting target ions in a sample, comprising a novel ion-selective ionophore, and to sensors containing said imprinted polymers.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Carrier-based ion-selective electrodes (ISEs) and optical sensors (optodes) have been used for detecting target ions in body fluids for many years. ISEs produce a measurable electrical change upon contact with a fluid sample containing target ions. Optodes, thin film ion-specific optodes and particle-based optodes typically contain a target ionophore, i.e., a lipophilic complexing ligand capable of reversibly binding ions, and an indicator ionophore. The target ionophore complexes with the target ion when present, and the indicator ionophore provides an indication of such complexing, such as by a color change.
The selectivity of the sensor is related to the equilibrium constant of the exchange reaction of target and interfering ions between the organic and aqueous phases. It strongly depends on the ratio of complex formation constants and stoichiometry of these ions with the ionophore in the membrane phase (1). The stoichiometry between the ionophore and ions is a very important issue for designing ionophores and optimizing the response and selectivity of the sensor because the changes in the stoichiometric ratio can influence the selectivity of the ionophore dramatically. With the development of host-guest chemistry, a large number of ionophores for different inorganic and organic ions have been synthesized.
For example, the oxapentanediamide type calcium ionophore ETH 129 complexes with Ca2+ with 3:1 stoichiometry and it also form Mg2+ complex with 2:1 stoichiometry (2, 3). The structures of these two complexes have been reported (2). In the ETH 129-Ca2+ complex, the nine oxygen atoms form a cavity with a radius of 102 pm, which is the ideal size for Ca2+ (radius, 106 pm). On the other hand, the coordination of Mg2+ is not optimized, therefore, ETH 129 has high calcium selectivity over magnesium ion and becomes one of the best ionophores for calcium ions. The high selectivity of ETH 129 based PVC-DOS and PVC-NPOE membrane has been reported by unbiased selectivity measurements (4).
Miniaturized optodes that function in accordance with bulk extraction principles have typically been either fiber optic or particle-based. Optical fiber-based optodes are usually fabricated by immobilizing a sensing layer on the distal end of an optical fiber by a simple dip-coating procedure. Sensors of this type have been developed for several clinical analytes, including H+ (5), Cl− (6), Na+ (7), and K+ (8). Although this approach offers the advantages of reduced sample volume and high signal-to-noise ratio, it does not appear feasible for multiplexed analysis.
Particle-based optodes have been produced by several different approaches, such as heterogeneous polymerization techniques (9–10), solvent casting (11), and very recently with a high-throughput particle generator (12) (13). An obvious advantage of particle-based optodes is their ability to independently interrogate a sample and produce a distinguishable analytical signal. To date, particle-based optodes have been used for very innovative applications, including flow cytometry (13) and intracellular monitoring (9), (14). The lifetime of these sensors, however, still remains a concern. However, for particle-based probes used for intracellular measurements, lifetimes have been reported as short as 30 minutes (9), thus validating the need for methods that improve sensor lifetime.
Traditionally, poly(vinyl chloride) (PVC) has been the polymer matrix most commonly used in membrane-based ISEs and hydrophobic bulk optodes (15). This is primarily due to its high tensile strength, chemical inertness, and plasticizer compatibility (10).
There are several disadvantages, however, associated with the use of plasticized PVC in ion-selective sensors, one of which is plasticizer leaching (17). For example, it is known that exudation of plasticizer and leaching of dissolved ionophores may ultimately limit the lifetime of carrier-based sensors. The former process may lead to a decreased solubility of the active sensing components (18), and the latter a loss of selectivity. For in vivo applications, where biocompatibility is essential, it has been found that such component leaching induces a serious inflammatory response (19). Further, in recent years ionophore-based chemical sensing systems have started to be drastically miniaturized in size, and the leaching of components is a much more significant problem in these cases.
One approach addressing the issue of diffusion that has received a substantial amount of effort is the fabrication of plasticizer-free polymers. Several such polymers have been evaluated in ISEs or ion-selective field effect transistors (ISFETs), including polyurethanes (20), polysiloxanes (6), silicone rubber (21), (22), polythiophenes (23), epoxyacrylates (24), and methacrylic (25) and methacrylic-acrylic copolymers (26), (27). Polymers synthesized via free radical initiated mechanisms, such as methacrylic-acrylic copolymers, appear quite attractive because of the numerous polymerization methods and infinite monomer combinations available to create polymers with a diverse range of physical and mechanical properties (28). Hall et al. have reported a substantial amount of work in this area (27, 28, 29). Particularly, in PCT application WO 00/54039, Hall et al. describe a selective polymer material with an acrylate backbone and a plurality of pendant lipophilic plasticising groups. Acrylate monomers are used to synthesize the polymers. The polymers are self-plasticising and thus are plasticizer free.
U.S. patent application Ser. No. 10/313,090 describes a plasticizer-free ion-detecting sensor comprising a copolymer of methacrylate monomers with R1 and R2 pendant alkyl groups, and an ionophore for detecting the target ion, where R1 is any of C1-3 alkyl group and R2 is any of C4-12 alkyl group. The use of methacrylate monomers of different pendant alkyl groups allows one to achieve a polymer material with not only a plasticizer-free effect but also a better mechanical strength for a desired Tg. In addition, methacrylate polymers of the present invention are less sticky and therefore much easier to handle. Furthermore, the methacrylate monomers of the present invention have less of a characteristic smell, and thus are much easier to manufacture.
Another approach to improving detection limits by reducing ion diffusion efficiencies across ion-selective membranes has been to covalently immobilize the ionophores to the membrane. Puntener et al. have recently reported a lead-selective ionophore and a hydrogen ion-selective ionophore covalently attached onto a polyurethane polymer that was then blended with traditional PVC-DOS (30). While the resulting sensing characteristics were very promising, the PVC membrane matrix significantly reduced the ion selectivity (30). Therefore, there remains a need for improved polymer materials, especially with respect to sensors comprising immobilized ionophores.
Kimura has reported using the sol-gel technique for immobilizing ionophores onto polymer materials. While this method has been successfully for ion-selective electrode applications, it remains untested for optical sensing strategies (31). Alternatively, two other immobilization methods have been reported. One approach directly grafts the ionophore onto an existing polymer with active sites (32, 33), while another method blends two different polymers together, with one of them containing the grafted ionophore (34). While these approaches offer practical approaches for ionophore immobilization, they are quite cumbersome to perform and typically require the presence of a plasticizer.
Recently, two hydrophilic crown ether-type K+-selective ionophores, 4-acryloylamidobenzo-15-crown-5 (AAB15C5) and 4′-acryloylamidobenzo-18-crown-6 (AAB18C6) (35), as well as a Na+-selective ionophore, 4-tertbutylcalix[4]arene tetracetic acid tetraethyl ester (27), have reportedly been polymerized with other monomers by a simple one-step polymerization method. These polymers containing grafted ionophores showed comparable selectivity and improved lifetime when compared to ISEs with free, unbound ionophore present.
However, in contrast to Na+ and K+ ionophores, immobilization of Ca2+ ionophores into an ion-selective electrode or optical sensor matrix has never been reported. Rosatzin has disclosed optical calcium sensors utilizing a covalently immobilized H+-selective chromoionophore in addition to a freely dissolved calcium ionophore (36). Response times were shown to be prolonged by the addition of the immobilized chromoionophore, but other sensing characteristics were satisfactory. However, all calcium bulk optodes reported so far utilize calcium ionophores that form very strong complexes with calcium (37). The resulting optode response ranges are therefore not yet suitable for calcium determinations in physiological samples at neutral pH.
Molecular imprinting (MIP) is a technique used to create selective molecular recognition sites in highly stable synthetic polymers (38). In this technique a target molecule to be imprinted is combined with a mixture of functionalized and non-functionalized monomers to form a complex, and then the complex is polymerized with a cross-linker. After the polymerization is complete the template molecules are removed and the resulting polymer matrix contains imprinted binding sites or “cavities” which are the “negative” of the imprint molecule. These cavities have the memory of the binding properties and size of the template molecules so they can rebind with the target molecules in a sample with high selectivity.
Most imprinted polymers are based on acrylic and vinyl monomers. By far the most used systems are matrices based on methacrylate, methacrylamide and styrene.
Imprinted polymers are usually divided into three types, including small spherical particles with size below micrometer (microspheres), thin layers, and surface imprints (39). Microspheres can be prepared by emulsion polymerization (40) and by precipitation polymerization (41, 42). Precipitation polymerization is similar to bulk polymerization except for the larger volume of solvents, such that the imprinted particles precipitate instead of polymerizing together to form bulk polymer. This method avoids the steps of grinding and sieving, which can result in the loss binding sites in the polymers (42).
Imprinted polymers are especially useful for making sensors. Because molecular imprinting polymers are highly crosslinked, the most common form of these polymers is a powder, which makes them very suitable for solid state extraction, binding arrays, and preparation of a stationary phase in for chromatography.
Some MIP sensors use optical transducers such as fluorescence (39). However, MIP sensors based on electrochemical detection are more attractive in terms of the simplicity of the devices and predominance of the electrochemical biosensors in the market (43). Another type of electrochemical sensor is potentiometric sensor.
Rosatzin et al. (44) reported the preparation of calcium ions and magnesium ions imprinted polymer from N,N′-dimethyl-N,N′-bis(4-vinylphenyl)-3-oxapentanediamide, divinylbenzene and styrene, however, only the dissociation constant (K) in methanol-water was determined for the polymers. The imprinted polymers prepared against calcium ions and magnesium were found to bind calcium ions 6 and 1.7 times greater, respectively, compared to the blank polymer without templates.
Although molecular imprinting has wide applications in sensing, most analytes are neutral organic compounds, and ion sensors based on molecular imprinted polymers have received much less attention. Because ion sensing must be performed in solution, mass sensitive devices cannot be used.
Carrier-based ion-selective electrodes (ISEs) and optical sensors (optodes) may also include ion-exchangers for improving their ion selectivities. However, loss or ion-exchangers from optodes is crucial to their viability because they typically respond via coextraction or ion-exchange equilibria, whereby a decrease in the concentration of sites results in a decrease in sensor sensitivity. Ion-exchanger leaching may also soon play a role in dictating the lower detection limit of ISEs. Thus, the development of more robust alternatives to the tetraphenylborates that exhibit improved stability and lipophilicity is warranted.
Tetraphenylborate derivatives have been used as ion-exchangers in cation-selective solvent polymer membrane electrodes and bulk optodes for many years. In addition to reducing anion interference, tetraphenylborates also decrease membrane resistance (45), and improve ionophore selectivity by stabilizing the concentration of ion-ionophore complex (46). The delocalized monoanionic charge that these compounds possess, in combination with their sterically hindered molecular structure make them very weakly coordinating. This is a characteristic that leads to weak, non-specific ion pair formation and maximum ionophore-mediated selectivity of the membrane (47).
Because the unsubstituted tetraphenylborate (TPB−) is susceptible to decomposition via acid hydrolysis, oxidants, and light, the search for more chemically stable derivatives began many years ago (46), (45), (48). One successful derivative is the highly substituted 3,5[bis-(trifluoromethyl)phenyl]borate (NaTFPB) (47). Even though halogenated derivatives, such as NaTFPB, are more lipophilic and more resistant to phenyl cleavage, acid hydrolyzed decomposition still occurs albeit at a much slower rate (47), (49). This shortcoming limits the use of tetraphenylborates in systems requiring an acidic sample pH, as in the case of heavy metals, such as Pb2+ (50).
Compounds that may be suitable alternatives to tetraphenylborates are carboranes, specifically closo-dodecacarboranes. These compounds possess many characteristics that may make them suitable ion-exchangers. Very weak ion pair formation is observed due to the lack of electron lone pairs and π-electrons, a property rarely found in anions (51). The desired lipophilicity of this class of carboranes can easily be tailored both at the boron vertices (48), (52–54) and at the carbon vertex (53), (54). The most lipophilic derivatives that have been synthesized are those of the perhalogenated (55) and peralkylated dodecacarborane anion (52).
U.S. patent application Ser. No. 10/313,090 describes the use of halogenated carboranes such as trimethylammonium-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 undecabromocarborane (TMAUBC) undecachlorinated (UCC), hexabrominated (HBC) and undecaiodinated (UIC) carborane anions as ion exchangers in ISEs and optodes.
In addition to potentially unparalleled lipophilicity, the carboranes possess many other characteristics that make them suitable for electrochemical applications. For example, they are not susceptible to acid and base hydrolysis and they are relatively inert to electrochemical oxidation (about 2.0 V vs. ferrocene/ferrocenium at Pt in dichloromethane) (51). High Ih symmetry and tangentially delocalized σ-bonding make the carboranes one of the most chemically stable classes of compounds in chemistry. Furthermore, their bulky size (nearly 1 nm in diameter) and sufficient charge delocalization meet the criteria imposed for sufficient ion exchanging. Another advantage, important for bulk optode studies, is their lack of absorption in the UV-Vis spectrum.